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Polybacterial infection of the amniochorion membranes: temporal regulation of microbial interaction and inflammatory/oxidative response

Grant number: 19/17279-4
Support type:Regular Research Grants
Duration: May 01, 2020 - April 30, 2022
Field of knowledge:Health Sciences - Medicine - Maternal and Child Health
Principal Investigator:Márcia Guimarães da Silva
Grantee:Márcia Guimarães da Silva
Home Institution: Faculdade de Medicina (FMB). Universidade Estadual Paulista (UNESP). Campus de Botucatu. Botucatu , SP, Brazil
Assoc. researchers:Jossimara Polettini ; Vera Lúcia Mores Rall

Abstract

Introduction: Although the etiology of preterm delivery is multifactorial, several microorganisms have been associated with its pathogenesis since the bacterial species most frequently isolated in the amniotic fluid of patients with intra-amniotic infection are commonly found in the vaginal microbiota. The infection of the amniotic cavity is the main cause of spontaneous preterm delivery, there is a complexity in this infection, since it is not a single entity, it has different bacterial communities involved, different interactions between species, different response to antibiotics and different clinical results. Objective: To evaluate the temporal regulation of the innate immune response in in vitro models of polybacterial infection of the amniochorion membranes with different associations of bacterial species associated to the preterm delivery, in the presence or not of different Lactobacillus species. Methodology: In each subproject a total of 10 amniochorion membranes obtained from term pregnancies will be challenged in vitro with different combinations of the main bacterial species identified in the lower genital tract of pregnancies complicated by preterm delivery in the concentration of 103 or 106 Colony- Forming Unit. Samples of amniochorion membranes without bacterial stimulation will be used as negative control and as a positive control will be used the lipopolysaccharide from Escherichia coli. For the evaluation of the regulation of expression and production of cytokines and receptors and oxidative stress biomarkers the supernatants and remaining tissues will be collected and stored at -80ºC at the following times: 6 hours, 12 hours, 18 hours and 24 hours. The supernatants obtained from the amniochorion membrane cultures will be submitted to protein quantification by Luminex panel technology composed of IL-1², IL-6, sIL-6R, sgp130, IL-8, IL-10, IL-13, GM-CSF, TNF-±, hBD1, hBD2, hBD3, PTX3 e PGE2. The remaining tissues will be used for the analysis of the gene and protein expression of mIL-6R, gp130, TLR-2, TLR-4, TLR-6, NOD1 and NOD2 by real-time PCR and Western Blotting, respectively. Additionally, immunolocalization with 3-dimensional imaging of IL-6 and its membrane receptors (mIL-6R and gp130) will be performed on the fetal membranes by immunofluorescence. To assess oxidative stress, after protein extraction, the levels of 3-nitrotyrosine, carbonylation and total antioxidative capacity will be quantified. The ultrastructural characterization of fetal membrane fragments after stimulation with different combinations of bacterial species will be performed by electron microscopy. In addition, the bacterial interactions in the fetal membranes will be analyzed by confocal microscopy. (AU)